Central to the replication of HIV-1 is the precursor Gag polypeptide (p55) which is able to direct the assembly and release of virion-like particles even in the absence of all other virus encoded components. The p55 thus contains all of the necessary signals involved in directing its intracellular transport from its site of synthesis in the cytoplasm to the plasma membrane where virus formation and budding occurs. Evidence indicates that this intracellular assembly is a multi-step process controlled by several unique Gag structural domains. We have used subcellular fractionation of cells expressing Gag mutant proteins to identify structural features involved in p55 intracellular transport, membrane binding, and/or function. One of these involves sequences within the Gag capsid domain (p24) which appear to be required for the association of p55 with membrane and/or cellular components which are insoluble in non-ionic detergents. Such interaction(s) may be essential for p55 subcellular targeting, membrane association, and/or viral budding. Other p55 signals include the co-translational N-myristoylation by the host N-myristoyltransferase and post-translational phosphorylation by protein kinase C which appear to be required for the subcellular targeting and/or membrane association of the mature Gag matrix protein (p17). A role for Gag phosphorylation in virus assembly is consistent with the inhibition of protein kinase C mediated phosphorylation of p55 and the parallel inhibition of Gag particle release from cells with the compounds Park-Davis 654077 and 668367. The role of these signals in regulating the interaction of Gag proteins with specific subcellular components (e.g., cytoskeleton, plasma membrane) has been the objective of these studies.